Antibiotic susceptibility of bacterial colonies: An assay ... · used to explore the efficacy of...

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Antibiotic susceptibility of bacterial colonies: An assay and experiments with

Staphylococcus aureus.

Xinxian Shaoa, Justin Kimb, Ha Jun Jeongb, Bruce Levin b,#

a Department of Physics, Emory University, Atlanta, Georgia, USA

b Department of Biology, Emory University, Atlanta, Georgia, USA

Running Head: Antibiotic susceptibility of bacterial colonies

#Address correspondence to Bruce Levin, [email protected].

Abstract:

A method is presented to evaluate in vitro the efficacy of antibiotics to treat bacteria

growing as discrete colonies on surfaces and the contribution of the colony structure to

the antibiotic susceptibility of bacteria. Using this method, we explored the relative

efficacy of six bactericidal and three bacteriostatic antibiotics to inhibit the growth and

kill Staphylococcus aureus colonies of different sizes, densities and ages. As measured

by the reduction in viable cell density relative to untreated controls, of the bactericidal

drugs tested ciprofloxacin and gentamicin were most effective. By this criteria,

ampicillin was more effective than oxacillin. Daptomycin and vancomycin were virtually

ineffective for treating S. aureus growing as colonies. The bacteriostatic antibiotic tested,

tetracycline, linezolid and erythromycin were all able to prevent the growth of S. aureus

.CC-BY-NC-ND 4.0 International licensenot peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was. http://dx.doi.org/10.1101/075515doi: bioRxiv preprint first posted online Sep. 16, 2016;

http://dx.doi.org/10.1101/075515

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colonies and did so even more effectively than daptomycin, which is highly bactericidal

in liquid culture. The results of these experiments and other observations suggest that

relative inefficacy of oxacillin, vancomycin and daptomycin to kill S. aureus in colonies

is due to the density and physiological state of the bacteria rather than the inability of

these drugs to penetrate the colonies. The methods developed here are general and can be

used to explore the efficacy of antibiotics to treat bacteria growing in biofilms as well as

discrete colonies.

Introduction

The rational (as opposed to purely empirical) approach to the design of antibiotic

treatment regimes is based on estimates of the changes in the serum concentration of the

drugs following their administration, pharmacokinetics (PK), and the relationship

between the concentration of the drug and the rates of growth and death of the target

bacteria, pharmacodynamics (PD) (1). Almost all we know about the PDs of antibiotics

and bacteria is from in vitro studies of planktonic cells maintained in well-agitated liquid

cultures (2-5) and the theoretical analog of these culture conditions, mass-action

mathematical models (6-9). Under these conditions all the bacteria in a population have

equal access to each other as well as resources, wastes, and allopathic agents like

antibiotics.

In the real world of infections, bacteria are more likely to live in physically structured

habitats, embedded in polysaccharide matrices known as biofilms (10-12) or as discrete

colonies on the surfaces of tissues or within semi-solids (13-15). Under these conditions,

the individual cells of bacterial population vary in their access to each other, nutrients,


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wastes and antibiotics. How does this reality of physically structured habitats affect the

pharmacodynamics of antibiotics and thereby the rational design of antibiotic treatment

protocols?

This question has been addressed and answers have been obtained for biofilms (11, 16-

22). In this report, we consider the pharmacodynamics of antibiotics and bacteria

growing as discrete colonies. We present a method to quantitatively evaluate the

antibiotic susceptibility of bacteria growing as colonies on surfaces and compare their

susceptibility to planktonic bacteria of the same densities and physiological state. Using

this method, we explore the susceptibility of Staphylococcus aureus Newman colonies of

different ages and sizes to six bactericidal and three bacteriostatic antibiotics. The

results of our study indicate substantial variation in the efficacy of the tested antibiotics to

treat S. aureus maintained as colonies. Antibiotics that are effective in killing S. aureus

in liquid culture are virtually ineffective when these bacteria are growing as colonies.

Material and Methods

Media. The Mueller-Hinton II (MHII) medium used in liquid and agar cultures was

Cation Adjusted MHII Becton Dickinson, (Franklin Lakes, NJ, USA).

Lysogeny Broth (LB) Becton Dickinson, (Franklin Lakes, NJ, USA) was used to prepare

LB agar plates for sampling.

Bacteria. Staphylococcus aureus Newman (generously provided by William Shafer) was

used in this investigation. This strain has a clinical origin, remains virulent and has been

extensively used in studies of staphylococcal pathogenicity (23).

MIC Estimation. MICs were estimated in vitro using the serial dilution procedure

similar to that described by (24) with modifications; we obtained these estimates in a


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standard MHII broth with inoculates of 5×107cells/ml as well a the standard

5×105cells/ml. In our serial dilution plates, we also used different starting concentrations

of the antibiotics to obtain more precise estimates of the MICs. The results are shown in

Table 1.

Procedure for the Colony Assay for Antibiotic Efficacy and Liquid Culture Controls.

A diagram of the method used to prepare the colonies, expose the bacteria to antibiotics

and estimate viable cell densities is presented in Fig. 1.

1- Establishing the colony and liquid cultures: Overnight cultures of S. aureus Newman

were grown in MHII and serially diluted in 0.85% saline. Using glass rods, the noted

numbers of bacteria were spread onto 25mm diameter 0.45 micron TuffrynTM filters. The

filters were placed onto 3 ml of 1.6% agar with 0.1% concentration of standard MHII (2.2

rather than 22 grams per liter) in the wells of Costar Macrotiter 6-well plates. In parallel,

the same densities of cells from the overnight culture were put into 9ml 0.1X MHII liquid

broth. The filters and the parallel liquid cultures were incubated at 37°C for 24 or 48

hours, the latter with shaking.

2- Exposing the bacteria to antibiotics: (i) For the experiments examining the effects of

antibiotics on intact colonies, filters with 24 or 48 hour old colonies grown on 0.1X MHII

(2.2 grams per liter) were transferred onto 3 ml of 1.6 agar media containing either

standard 1X MHII or 3X MHII (respectively 66 grams per liter) with either 10X or 40X

MIC of the antibiotic as well as an antibiotic-free controls. The colony cultures were

incubated at 37°C for an additional 24 hours. (ii) For the experiments with planktonic

cells growing in liquid the 24 or 48-hour cultures in 0.1X MHII were diluted to the

approximate density of bacteria in the corresponding colony cultures and then put into


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flasks containing either 1X MHII or 3X MHII medium with 10X MIC of the antibiotics

or antibiotic-free controls. These liquid cultures were incubated for 24 hours with

continuous shaking. (iii) For the bacteria dispersed from the colonies, the filters with 24

or 48 hour old colonies were transferred into 3 ml of 3X MHII broth with 10X MIC

ciprofloxacin (DIS-CIP) or oxacillin (DIS-OXY) or without antibiotics (DIS-CON).

These cultures were then incubated at 37℃ with continuously shaking for 5 or 24 hours.

3- Sampling and estimating the viable cell densities: The liquid and the dispersed colony

cultures were sampled directly by serial dilution and plating on LB agar. The filters with

colonies were removed from the agar, placed in 10ml of 0.85% saline and the cells were

washed and suspended in saline with plastic Pasteur Pipettes. To suspend the cells and

break-up clumps the saline containing filters and cells were sonicated (Bronson, 2510R-

DTH, output 100W, 42 kHz 6%) for 5 minutes and vortexed for 10 seconds before serial

dilution.

RESULTS

The growth of S. aureus Newman in liquid and as colonies. The pharmacodynamics of

antibiotics and bacteria depend on the physiological state of the cells, which is reflected

in their growth rates and the densities of their population (25). To explore these effects

and determine the contribution of the physical structure of the population the growth of S.

aureus, we estimated the densities of cultures growing as colonies with that of cultures of

the same initial density growing in liquid (Fig. 3).

In liquid culture with 0.1X MHII there is no significant effect of the initial inoculum on

the viable cell density of S. aureus Newman at 24 hours (P ~ 0.9) (Fig. 2A). On the other


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hand, with 3X MHII in liquid, at all inoculum densities the bacteria are still growing at 24

hours. At 48 hours in liquid there is no effect of the inoculum density in either 0.1X

MHII or 3X MHII (P>0.05) (Fig. 2A and C). As colonies, at all inoculum densities, the

population is still growing at 24 hours on both 0.1X MHII and 3X MHII agar (Fig. 2B

and D). At 48 hours in both 0.1X MHII and 3X MHII the population inoculated with 50

cells and thereby larger colonies is still growing, whilst those inoculated with 103 and 104

cells appear to be at stationary phase (p>0.05).

We interpret these results to mean that when liquid cultures in 0.1X MHII are exposed to

antibiotics, they are already at stationary phase at 24 hours, but in 3X MHII they are still

growing. At 48 hours with inoculates of 103 and 104 cells, these colonies are no longer

growing and presumably the cells are at stationary phase, which is not the case for

cultures initiated with 50 colonies. It should be noted, that at stationary phase the

density of S. aureus growing as colonies on 0.1X MHII agar is significantly greater than

that in the corresponding liquid culture (p<<0.05 for 103 C and 104 C) (also see (26) ) for

similar observations). This is not the case for the richer media, 3X MHII, where there is

no significant difference in the 48 hour estimated densities when the bacteria are growing

in liquid or as colonies (p>>0.05).

The effect of physical structure on the susceptibility of colonies to antibiotics. We

initiate our consideration of the relative antibiotic susceptibility of S. aureus growing as

colonies and liquid with two bactericidal antibiotics, ciprofloxacin and oxacillin. To

determine the contribution of the physical structure of the colonies to these antibiotics,

we compare the extent of antibiotic-mediated killing of S. aureus in colonies with that of

planktonic cells released from colonies (dispersed, DIS) and liquid cultures of the same


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age as the colonies (LIQ). All were treated with 3X MHII broth or agar containing 10X

MIC of these drugs and the viable cell densities were estimated after 5 and 24 hours of

exposure to the antibiotics. In Fig. 2, we illustrate the experimental setup and the effects

of exposure on the size of 48-hour colonies in the absence of antibiotics and following

exposure to oxacillin and ciprofloxacin.

For this assay, we estimated the viable cell density of S. aureus at 5 and 24 hours of

exposure to oxacillin and ciprofloxacin of cells maintained in colonies (COL), cells

released from colonies (DIS) and cells maintained in liquid cultures (LIQ) of the same

age (Fig. 4). As noted earlier, both the 24-hour and 48-hour control cultures (CON)

continued to increase in density after being transferred to the richer media (Fig. 4) and the

size of colonies and the pigmentation dramatically increased (Fig. 2). For ciprofloxacin,

24-hour old planktonic cells grown in liquid culture or released from colonies were more

susceptible to killing by this antibiotic after 5 hours of exposure than those maintained in

colonies. After 24 hours of exposure, the bacteria within colonies or dispersed from

colonies appeared more sensitive to killing by this fluoroquinolone than those in liquid

culture (p<< 0.05). After 48 hours of growth, the bacteria within colonies are more

refractory to ciprofloxacin than they are as planktonic cells. For both the 24 and 48-hour

cultures, relative to the antibiotic-free controls, ciprofloxacin was effective in preventing

the growth and killing S. aureus with colonies.

Oxacillin was clearly less effective than ciprofloxacin in killing S. aureus in planktonic

cells as well as within colonies. Moreover, there appeared to be little difference in the

efficacy of this beta-lactam antibiotic in killing S. aureus in colonies relative to that of

planktonic cells in liquid. This experiment was repeated three times and similar results


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obtained (data available upon request).

Effect of colony size and density on the extent of antibiotic-mediated killing. How

does the size (number of cells within and the physical dimensions) of colonies affect their

susceptibility to killing by antibiotics? How does the distance between colonies, the

density on the agar affect their susceptibility to killing by antibiotics? Most importantly,

how effective are different antibiotics in inhibiting the growth and killing of S. aureus as

planktonic cell liquid and within colonies. To address these questions, we prepared

filters with ~ 50 cells and ~ 104 cells, respectively large, widely dispersed colonies and

small, densely distributed colonies. These filters were placed on 0.1X MHII and grown

for 24 or 48 hours, at which time they were placed on 1X MHII agar containing 10X MIC

of one of six bactericidal antibiotics or one of three bacteriostatic drugs.

In Table 2 we present the results of these experiments with cultures initiated with

approximately 50 colonies. In the absence of antibiotics in both liquid and as colonies,

the bacterial population increased in density. The bacteriostatic drugs, tetracycline

erythromycin and linezolid prevented the population from growing in colonies as well as

in liquid. The most effective bactericidal antibiotic in killing both planktonic S. aureus

and with within colonies was ciprofloxacin. For the younger, 24 hour cultures this

fluoroquinolone was more effective in killing the bacteria in colonies than in liquid, the

opposite was true for the older 48 hour cultures. For the younger cultures gentamicin

was similarly effective in killing the cells in colonies as it was when these bacteria were

in liquid. This is not the case when the cultures were 48 hours old at the time of exposure

to this drug, which was ineffective in killing these bacteria when they were in colonies.


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Vancomycin was moderately bactericidal in liquid but totally ineffective when bacteria

were in colonies. At these densities, daptomycin was effectively bacteriostatic in both

liquid and younger colonies, but failed to prevent the growth of S. aureus the 48hour old

colonies. This may be well attributed to the relatively high initial density of the culture

and the degradation of this drug at this higher cell densities (27, 28). The beta-lactam

antibiotics were less effective in killing S. aureus in colonies than they were when these

cells were in liquid.

In the cultures inoculated with ~104 cells the colonies were much smaller and more

crowded than the colonies inoculated ~ 50 cells per filter (Table 3). Of the 9 antibiotics

considered, gentamicin and ciprofloxacin were the most effective at killing S. aureus

when treating the 24-hour cultures, even more effective than they were with the larger

colonies. Apart from gentamicin and ciprofloxacin, all the other antibiotics were less

effective comparing to corresponding cultures inoculated ~ 50 cells per filter. The

bacteriostatic drugs inhibited the growth of both 24 and 48 hour colonies but failed to do

so in all liquid cultures. Vancomycin failed to kill the bacteria in all cases. In general, the

48-hour crowded colony cultures were the most refractory to antibiotics.

Discussion

In designing and performing these experiments we had two goals. One, to develop and

evaluate a facile and broadly applicable method for quantitative studies of the antibiotic

susceptibility of bacteria growing as colonies. Two, to apply this procedure to explore

the efficacy of different antibiotics to inhibit the growth and kill Staphylococcus aureus

in colonies. While there have been a number of studies of the efficacy of antibiotics for

treating bacteria in biofilms (17-22, 29-32), to our knowledge this is the first


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investigation to explore antibiotic-mediated inhibition of replication and killing of

bacteria growing as discrete colonies on surfaces.

The procedure developed here can be applied to virtually any bacteria that when grown in

vitro forms colonies. This same procedure could be employed to evaluate the

susceptibility of single and multi-species biofilms to antibiotics. For this, instead of

seeding the filters with relatively few bacteria to form discrete colonies, the filters could

be seeded with large numbers of bacteria of the same or multiple species. The sampling

methods would be identical to those described in here. By comparing liquid cultures and

bacteria released from colonies (or biofilms) of the same density and stage of growth (age)

this method provides a way to evaluate the contribution of the physical structure of the

population to its susceptibility to antibiotics.

The results of this study indicate that there is substantial variation among bactericidal

antibiotics in their efficacy for killing bacteria within colonies. Of the six bactericidal

antibiotics considered here, ciprofloxacin was most effective, followed by gentamicin.

Of the beta-lactam antibiotics, ampicillin was more effective in killing S. aureus in

colonies than oxacillin. In our experiments, daptomycin, which is considered bactericidal

(33-35), was no more capable of killing S. aureus in colonies than the antibiotics that are

deemed bacteriostatic, tetracycline, erythromycin and linezolid. Indeed, this cyclic

peptide was less effective in preventing the proliferation of S. aureus in more mature (48

hour) colonies than these bacteriostatic drugs. Vancomycin, which is commonly

employed for treating methicillin resistant S. aureus (7, 9, 32, 34, 36), was virtually

ineffective for either preventing the replication of or killing S. aureus Newman in

colonies.


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The preceding conclusions about the relative efficacy of the different antibiotics for

treating S. aureus as colonies is based on a common dose of 10X MIC of these drugs with

24 and 48-hour inoculum densities respectively of ~2×107 and ~2×108 cells per ml for the

experiments with 50 colonies and ~3×108 and ~9×108 for the experiments initiated with

104 colonies. These densities are substantially greater than the recommended 5x105 cells

per ml for estimating MICs (24) by serial dilution. To some extent the differences in

relative efficacy of the bactericidal antibiotics to kill S. aureus in colonies may be

attributed to a density (inoculum) effect (27). On the other hand, we don’t see the utility

of reducing the density of cells treated to make these drugs more effective in this

experimental system. From a clinical perspective, concern is to treat established

infections the densities of which are likely greater than the 5×105 (37, 38).

What about increasing the dose of the antibiotics that were ineffective for treating

colonies in these experiments? We have explored this possibility with 40X MIC of

oxacillin and vancomycin. The results of these experiments suggest that even at these

high concentrations, these antibiotics are ineffective for treating S. aureus Newman in

colonies (Table S1).

On first consideration it may seem that the relative inefficacy of vancomycin, oxacillin

and daptomycin to kill or prevent the replication of S. aureus in colonies may reflect the

inability of these drugs to diffuse through the membrane on which the colonies are

growing and then through the colonies. We do not believe this is the case for oxacillin or

vancomycin. Although it is known that to some extent biofilms reduce the rate of

diffusion of antibiotics. In our experiments, however, the time of exposure to these drugs

was relatively long, 24 hours. We expect that the diffusion effect would be small. Based


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on what is known about the diffusion rates of these drugs in Staphylococcus in biofilms it

seems reasonable to assume the bacteria within these colonies would have been exposed

to substantial concentrations of these drugs (30, 31, 39). Moreover, the cells dispersed

from colonies before treatment with these drugs were no more susceptible than those in

intact colonies, even though the bacteria were in liquid and confronted with the same

concentration of these antibiotics (Table S1). We suggest the primary reason for the

relative inability of these antibiotics to kill S. aureus in colonies can be attributed to the

density and physiological state of the bacteria, rather than the structure of the colonies.

One possible explanation for why daptomycin is not effective in colonies but is in liquid

is that its mode of action is thwarted by the structure of the colonies. It has been

proposed that daptomycin operates by depolarizing the cell membrane which results in

leakage of ions (7-9, 30). If, however, the cells are within colonies the ions lost by

individual cells would remain in the collective and thereby shared by all in this

community.

Acknowledgements

This work was supported by a grant from the US National Institutes of Health, GM

091875 (BRL) and Laney Graduate School of Emory University.

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Table 1 The MICs of 9 antibiotics in MHII broth of different concentrations and cultures

initiated with different numbers of viable cells, unit of the concentrations is in µg/ml.

Antibiotic Source

Inoculum Size

5x105cells/ml 5x107cells/ml

Vancomycin hydrochloride, Sigma 1.3 3.5

Gentamicin AppliChem 0.9 1.1

Ciprofloxacin AppliChem 0.3 0.3

Daptomycin TCI 1.3 4.0

Ampicillin Sigma 0.9 1.3

Oxacillin Sigma 0.2 0.5

Tetracycline* Sigma 0.5

Erythromycin* MP Biomedicals 0.5

Linezolid* Chem - IMPEX 1.1

* We have not included the effects of inoculation densities on the MICs of the

bacteriostatic antibiotics because the results of our experiments suggested that the density

of the exposed bacterial population contributes little to the susceptibility of colonies to

the action of these drugs.


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Figure 1 The experiment setup and protocol to grow and treat S. aureus Newman grown

as colonies on filters. The growth of bacterial colonies is the same for the colony cultures

and the dispersed cultures. After 24 or 48 hours of growth, the filters with bacterial

colonies are either transferred into fresh liquid broth for the dispersed cultures, or onto

fresh agar for the colony cultures.


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Figure 2 S. aureus Newman colonies grown on filters, roughly 50 colonies on each filter.

(A) Colonies on filters grown for 48 hours on 3 ml of agar media with 0.1X MHII. (B) 48

hour colonies placed on 3ml agar with 3X MHII for an additional 24 hours. (C) A filter

with 48 hour colonies placed on 3ml 3X MHII agar with 10X MIC oxacillin for an

additional 24 hours. (D) A filter with 48 hour colonies placed on 3ml 3X MHII agar with

10X MIC ciprofloxacin for an additional 24 hours.


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Figure 3 Density and time before resource saturation of liquid and colony populations of

S. aureus Newman. Cultures were inoculated with 50 cells, 103 cells and 104 cells

respectively. Viable cell density of S. aureus Newman at 24 (blue bar) and 48 (orange bar)

hours grown as (A) Planktonic cells in liquid culture with 0.1X MHII, (B) Colonies on

filters on agar with 0.1X MHII, (C) Planktonic cells in 3X MHII broth, and (D) Colonies

on filters on 3X MHII agar. Error bars are SEMs.


http://dx.doi.org/10.1101/075515


20

Figure 4 Relative survival of S. aureus exposed to 10XMIC ciprofloxacin (CIP) or

oxacillin (OXA), in liquid (LIQ), as planktonic cells suspended from colonies (DIS), and

as intact colonies (COL). An average of 50 colonies was inoculated on each filter. The

CON cultures are antibiotic-free controls. (A) Cultures grown for 24-hour before

exposure to the antibiotic. (B) Cultures grown for 48 hours before exposure to the

antibiotics. The viable cell densities were estimated at 5 hours and 24 hours of exposure,

respectively the blue and red bars. The dashed lines are the viable cell densities before

exposure to the antibiotics. Means and standard errors for three replicates.


http://dx.doi.org/10.1101/075515


21

Table 2 The results of experiment with S. aureus Newman cultures initiated with an

average of 50 cells per 3ml of MHII broth/agar containing 10X MIC of the drugs listed.

Cultures grew for 24 or 48 hours in 3ml of 0.1X MHII broth/agar before exposure to the

antibiotics in 1X MHII. The viable cell densities (CFU/ml) were estimated 24 hours

after exposure to the antibiotics.


http://dx.doi.org/10.1101/075515


22

Table 3 Antibiotic-mediated killing of S. aureus Newman cultures initiated with an

average of 1e4 cells per 3ml of MHII broth/agar containing 10X MIC of the drugs listed.

Cultures grew for 24 or 48 hours in 3ml of 0.1X MHII broth/agar before exposure to the

antibiotics. Viable cell density (CFU/ml) was measured at 24 hours after exposure.


http://dx.doi.org/10.1101/075515


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